Project Summary/Abstract Metastatic disease constitutes the vast majority of deaths associated with breast cancer, and therefore the ability of cancer cells to invade through the extracellular matrix and metastasize to distant locations represents a critical juncture in the pathogenicity of a tumor. Secretion of pro-invasive and pro-metastatic proteins is an important mechanism by which cancer cells signal to each other and with their surroundings to facilitate this behavior. Therefore, understanding the biology underlying cancer cell secretion is of great interest to the scientific and clinical cancer communities. Classically, secreted proteins containing a signal peptide are targeted to the endoplasmic reticulum (ER)-to-Golgi route for proper folding and packaging. However, both normal and cancer cells can also actively secrete proteins including pro-invasive factors through unconventional pathways that bypass the ER-to-Golgi route. Elucidating the molecular mechanisms behind the unconventional secretion of these and other as of yet unknown pro-invasive proteins has long-term implications for improved diagnosis, disease monitoring, and targeted therapy in cancer. Mounting genetic evidence indicates that a Golgi-associated protein called GRASP55 plays a critical role in unconventional secretion. However, the mechanism underlying GRASP55-dependent unconventional secretion and the biological significance of this pathway are largely unknown. To address these questions, the specific aims of this project are to 1) Determine the role of the ER-Golgi interface in GRASP55-dependent unconventional secretion and 2) Determine whether GRASP55 promotes non-cell autonomous invasive phenotypes of breast cancer cells in vitro. This project will utilize a combination of methods including mass spectrometry, secreted luciferase reporter assays, genetic manipulation of model and cancer cell lines, immunofluorescence microscopy, and in vitro cancer cell migration and invasion assays. Successful completion of this project will clarify the mechanisms underlying unconventional secretion, a basic cellular process, and elucidate its functional significance in cancer biology.